The elucidation of molecular alterations that occur during human breast cancer development may permit the identification of preventative strategies for women at high risk. Our goal is to identify proteins whose expression levels vary between surgical biopsies containing matched normal ductal/lobular units and Ductal Carcinoma in Situ (DCIS) lesions using laser capture microdissection (LCM) and proteomics. Ten sets of two-dimensional (2D) gels were evaluated, containing either matched whole sections from normal ducts or DCIS, or up to 10,000 LCM procured epithelial cells. Differential protein expression was confirmed by image analysis. 315 proteins spots were excised and sequenced by mass spectrometry. Fifty seven proteins were differentially expressed between normal ductal/lobular units and DCIS. Differences in overall protein expression levels and post-translational processing were evident. Ten differentially expressed proteins were validated in independent DCIS specimens, and 14/15 proteomic trends from 2D gel analyses were confirmed by standard immunohistochemistry using a limited independent tumor cohort. Many of the proteins identified were previously unconnected with cancer, including proteins regulating the intracellular trafficking of membranes, vesicles, cancer prevention agents, proteins, ions and fatty acids. Other proteomic trends related to cytoskeletal architecture, chaperone function, apoptosis, genomic instability and the microenvironment. Proteomic analysis of DCIS revealed differential expression patterns distinct from previous nucleic acid based studies, and identified new facets of the earliest stage of breast cancer progression.